1. Field of the Invention
The present invention relates to a method for treating individuals having inflammation or preventing inflammation in individuals at risk for inflammation, more specifically individuals with chronic inflammation as evidenced by elevated acute phase reactants including C-reactive protein and serum fibrinogen, elevated platelet count or platelet activity, elevated blood glucose, or any component or combination of components of the metabolic syndrome, from progressing to the natural outcome of the syndrome, such outcome being diabetes mellitus, coronary artery disease, and related complications of diabetes mellitus
2. Description of the Related Art
The distribution of fat characteristic of the Metabolic Syndrome (Syndrome X) (a precursor to a form of Type II Diabetes) resembles the lipodystrophy seen in longer term HIV Disease survivors, and which is also associated with insulin resistance. There is currently a debate as to whether the cause of lipodystrophy and abnormal glucose tolerance in HIV survivors is due to treatment with protease inhibitors, or to long-term survival with HIV infection. There is clearly continual antigenic stimulation in this situation. Chronic antigenic stimulation results in inflammation which, in turn, can result in abnormal immune function, cardiovascular disease, and other sequelae.
Earlier studies had described that Type II diabetes had a very strong familial (dominant) inheritance pattern (GOTTLIEB AND ROOT, DIABETES 17:693-704, 1968). Current Type II diabetes, associated with the Metabolic Syndrome, has not been reported to have such a familial association (SINHA, ET AL., N. ENGL. J. MED. 346:802-810, 2002).
Studies have shown an increase in coronary heart disease mortality in association with air pollution and increased diabetes mellitus in association with release of dioxins (for example, HENRIKSEN, G L, EPIDEMIOLOGY 8:252-8(1097)). C-Reactive Protein levels have been elevated in these studies. (Elevated C-Reactive Protein is a marker for an inflammatory response.) The frequency of obesity has been increasing markedly in all populations.
Therefore, chronic antigenic stimulation, whether by infection or environmental pollutants can overwhelm the innate immune system's ability to control (remove) these substances leading to uncontrolled inflammation, failure of insulin to affect liver and muscle enzymes to control blood glucose, leading to impaired glucose tolerance, hyperinsulinemia, insulin resistance, dyslipidemia, elevated triglycerides, and i.e. the Metabolic Syndrome.
This invention concerns the relationship between cell-mediated immunity and pathological conditions associated with cell-mediated immune dysfunction. Such conditions include HIV Disease and other chronic infectious diseases caused by particular pathogenic organisms, including HIV, gingivitis, candida sp., those caused by chronic inflammation resulting from exposure to environmental toxins and particulates, and those resulting from other stressors such as trauma and aging. The invention also concerns other conditions in which there is dysfunctional immunity resulting in metabolic and inflammatory conditions.
A typical manifestation of cell-mediated immunity is the delayed type hypersensitivity (“DH”) skin reaction. A DH skin reaction is observed when an appropriate antigen is injected intradermally. Within 24 to 48 hours, local inflammation (erythema) and a swelling and thickening (induration) are observed in a sensitive individual. The degree of sensitivity may be measured by the size and severity of the reaction. The DH reaction also presents characteristic histological findings—specifically, perivascular infiltration of leukocytes in the inflamed area. The cells seen at the site of a DH reaction are derived from the peripheral blood leukocyte population.
The mechanisms of cell-mediated immunity are as yet incompletely understood. It is known that the cells which mediate the response are capable of responding in a variety of ways to a challenge from an antigen. These responses include: proliferation of cells bearing specific sensitivity to a given antigen; the induction and multiplication of cells mediating a variety of immune functions, including antibody production; and reactions against foreign cells, tumors, and other foreign substances.
The present invention relates to the use of (1) endogenous regulators of the immune system, which are isolated from dialyzed extracts of leukocytes, and synthetic similar products; and (2) compositions containing the immunoregulators. These immunoregulators, whether produced endogenously by the human individual or provided exogenously as a therapeutic agent, profoundly affect the quality and quantity of cell-mediated immunity responses; and are useful in the treatment of clinical conditions characterized by inadequate or inappropriate reaction to antigens including, but not limited to HIV Disease, rheumatoid arthritis, sarcoidosis, and malignancy.
Earlier A. Arthur Gottlieb Patents: In A. Arthur Gottlieb U.S. Pat. No. 4,468,379, it was disclosed that endogenous materials exist that amplify the speed and magnitude of the cell-mediated immune system response. These amplifier materials are distinguished from so-called transfer factors in that amplifiers do not transfer to a subject an immune response to a mitogen or antigen to which the subject has not previously been exposed and is not concurrently exposed, while transfer factors are said to do so. Moreover, amplifiers nonspecifically increase cell-mediated immune system responses to mitogens and antigens to which the subject has previously been or concurrently is exposed, while transfer factors are specific to particular antigens.
The material designed “amplifier 1” in the '379 patent is now known by the inventor to be a mixture of various things. They include what is referred to subsequently in the present patent application as YG-material and what is referred to subsequently in the present patent application as YGG-material. It was suggested in A. Arthur Gottlieb U.S. Pat. No. 4,616,079 that amplifiers appear to act on T-helper cells (T4 cells) in a way that causes them to produce chemical mediators (lymphokines) whose effect is to increase the speed and/or magnitude of cell-mediated immune system response to antigens and other means of activating a cell-mediated immune system response. (The term “recall antigen,” as used hereinafter, refers to an antigen to which a subject has previously been exposed.) Indicia of this response include DH reaction to recall antigens, production of IL-2 and gamma interferon, and potentiation of cytotoxic cells.
It is known that various diseases and pathological conditions, such as HIV Disease (also referred to as Acquired Immune Deficiency Syndrome (AIDS) and AIDS-Related Complex (ARC)), as well as other infectious agents, chemotherapy, radiation, aging, other forms of physiologic and psychological stress, and environmental exposures depress the immune system response. As a result, there is increased susceptibility to opportunistic infections, malignancies, and other pathological conditions that a normal immune system would have confronted. Frequently (and for some conditions, invariably), the result is death. Administration of immunoregulators (referred to as “amplifiers” in other A. Arthur Gottlieb patents, including U.S. Pat. No. 5,100,663) provides a means of improving cell-mediated immune system responsiveness, where the cell-mediated immune system remains sufficiently intact for it to respond to such challenge.
Earlier A. Arthur Gottlieb patents describe means of extracting amplifier materials from human leukocyte dialysates by reverse-phase HPLC processes. A. Arthur Gottlieb U.S. Pat. No. 4,699,898, as well as in other related patent applications of the inventor, including U.S. Pat. No. 5,100,663, the inventor disclosed his discovery of peptide products containing Tyr-Gly (YG) and Tyr-Gly-Gly (YGG) amino acid residue sequences, that are immunologically active components in the partially purified dialysate fractions previously described in earlier A. Arthur Gottlieb patents, such as A. Arthur Gottlieb U.S. Pat. No. 4,616,079.
Earlier A. Arthur Gottlieb patents did not teach that the YG and YGG peptides had any effect on the consequences of chronic antigenic stimulation, including inflammation, inflammatory disease, metabolic alterations, or on regulation of indicators of such consequences including elevated C-Reactive Protein, fibrinogen, sialic acid, or blood glucose.
The earlier A. Arthur Gottlieb patents may also be consulted for other general background information on immunoregulators (the same as what are called immunoamplifiers, amplifiers, or immunomodulators in those patents) and their use.
Coy: Coy U.S. Pat. No. 4,127,534 describes tripeptides of the form Tyr-X-Gly, where X is a D-amino acid. Coy asserts that these products have analgesic and related utility, as indicated by rat tail flick or other tests; accordingly, they may be used as substitutes for such medications as aspirin and sedatives. Coy claims pharmaceutical compositions that contain a “therapeutically effective amount” of Tyr-X-Gly, including Tyr-D-Ala-Gly. Coy asserts in the body of his specification that a therapeutically effective amount of the product for purposes of the disclosed utility is from 0.001 mg per kg of bodyweight to 100 mg per kg of bodyweight, administrated daily. (Extrapolated to an 80 kg person, this amounts to a daily dose of approximately 0.1 mg to 10 g; 0.1 mg is equivalent to approximately 300 nanomoles, and 10 g is equivalent to approximately 0.03 moles.) It should be noted that the relevant language of Coy's specification is in the present tense, indicating use of prophetic examples. (No therapeutic examples are provided in the specification, and no statements about utility or dosage are made in the past tense.)
Coy does not assert any immunological use of the products. Coy does not describe any of the D-amino acid group as a means of preventing cleavage of the Tyr-Gly bond by endogenous enzymes. Coy does not describe any utility for doses of less than the aforesaid minimum daily amounts (0.001 mg/kg, 0.1 mg, and 300 nM).
Plotnikoff: Plotnikoff U.S. Pat. No. 4,537,878 discloses and claims the use of endogenous endorphins and enkephalins to stimulate the immune system. The dosage amounts actually used in vivo (Plotnikoff's Examples VIII to XI) were from 1 microgram (μg) per kg to 50 μg/kg, single i.v. dose. Elsewhere, however, Plotnikoff refers to a therapeutic dose of from 1 μg/kg to 30 mg/kg, and to a preferable dosage rate of from 0.01 fg/kg to 250 μg/kg. No explanation is given for the inconsistencies, and no data in the specification indicates a reason why these latter dosage rates were mentioned or claimed. (They do not appear in examples or similar data.)
The molecular species whose use Plotnikoff discloses are the endogenous enkephalin pentapeptides, and longer endorphin polypeptide extensions thereof (extended from the C-terminal end). Plotnikoff does not disclose use of any nonendogenous peptides, nor anything concerning use of dipeptides, tripeptides, or tetrapeptides. Plotnikoff does not indicate that Tyr-Gly or Tyr-Gly-Gly have any immunological or other utility. Plotnikoff does not show that any products, other than enkephalin, have utility in treating AIDS or ARC.
Schwartz: Schwartz et al., Biological inactivation of enkephalins and the role of enkephalin-dipeptidyl-carboxypeptides (“enkephalinase”) as neuropeptidase, ENKEPHALIN METABOLISM 29:1715 (1981), extensively reviews work that has been done in the field of enzymatic breakdown of enkephalins. Schwartz summarizes the paper as follows:
In this review it will be shown that enkephalins are rapidly hydrolyzed in vivo and that several peptidase activities have been identified which are able to cleave these molecules to give various biologically-inactive fragments. (Emphasis added.)
Schwartz et al. and the work summarized in the review teach that various endogenous enzymes cleave (hydrolyze) the Gly-Phe, Gly-Gly, and Tyr-Gly bonds of endogenous mammalian polypeptides, such as Leu-enkephalin and Met-enkephalin into what Schwartz alleges are “biologically inactive fragments.” Such fragments include what Schwartz refers to as Tyr-Gly, which in context apparently means a dipeptide containing Tyr and Gly amino acid residues, in that order. But Schwartz does not indicate what side chains or other groups, if any, are attached to the amino acid residues or what specific molecular structure is present in the Tyr-Gly product.
Schwartz and the work summarized in the review also disclose various means of inhibiting such enzymatic cleavage, including N-methylation of the Tyr residue; esterification, amidification, and alcoholation of the C-terminal carboxyl; insertion of a D-amino acid residue (such as D-Ala) into the chain near the C-terminal end; and mixture with bacitracin, puromycin, bestatin, amastatin, or thiorphan. (It is also known in pharmaceutical art, although not discussed in Schwartz, to bind or complex an enzyme-inhibiting agent to a therapeutically active molecule, so that the agent will preferentially bind to the active site on the enzyme that is to be inhibited, thereby preempting that site and thus keeping the enzyme from hydrolyzing the molecule to be protected. This is exemplified by the use of the product sulbactam, a beta-lactamase inhibitor used to protect ampicillin from beta-lactamase; thus, UNA-SYN.™. (Pfizer) is a mixture of sulbactam and ampicillin, while sultamicillin is ampicillin complexed or otherwise linked with sulbactam via an ester. It is also known, for example in the case of the synthetic penicillins, to introduce a large group (such as methyl) at a location on a therapeutically active molecule where there would otherwise be a space providing a site for enzyme attachment, which results in hydrolysis. The result of occupying such a space is to inhibit enzymatic degradation of the molecule thus protected.)
The Schwartz paper does not mention any immunological activity or other utility of the allegedly useless and biologically inactive fragments resulting from enzymatic action on enkephalins.
Delivery of drug via hydrolysis: It is known that a therapeutically active molecule may be delivered by administering to a patient a different molecule that hydrolyzes, as a result of the action of endogenous enzymes, to fractions that include the desired therapeutically active molecule. Perhaps hetacillin is the best known example. Hetacillin breaks down in the human body to ampicillin. A legal controversy ensued internationally, following the introduction of hetacillin, over whether the manufacture, use, and sale of hetacillin infringed patents on ampicillin.
Zacharie: Zacharie, et al. (J. MED. CHEM. 42:2046 (1999).) have confirmed the work of A. Arthur Gottlieb, as previously described, by testing Tyr-Gly and Tyr-Gly-Gly peptides for biologic activity in vitro. Additionally, Zacharie, et al. made covalent modifications to these molecules, as taught by A. Arthur Gottlieb, by thioacylating the molecules, thereby increasing biologic activity.
Kayser and Meisel: A. Arthur Gottlieb's teachings concerning the biologic activity of Tyr-Gly and Tyr-Gly-Gly have also been confirmed by Kayser and Meisel (FEBS LET. 383:18 (1996)) who show, by in vitro testing, that these peptides which can also be derived from the breakdown of certain milk proteins are immunologically active molecules.
Commercial Tyr-Gly: Tyr-Gly is sold as a chemical reagent (L-tyrosylglycine) by Sigma Chemical Co., St. Louis, Mo., among others. Tyr-Gly is not sold in U.S.P. grade, and it is illegal under applicable laws to sell Tyr-Gly for use as a pharmaceutical. Commercial grade Tyr-Gly is not considered free of pyrogens, endotoxin, and other pharmaceutically unacceptable constituents. The presence of such pyrogens, endotoxin, and other pharmaceutically unacceptable constituents makes a product unacceptable for use as a drug, as that term is defined by federal statute, both under generally recognized medical principles and under FDA regulations. To the extent of the inventor's knowledge, no pharmaceutical preparations of this product are or have been available.
Commercial Tyr-Gly-Gly: Tyr-Gly-Gly is sold as a chemical reagent (L-tyrosylglycylglycine) by Sigma Chemical Co., St. Louis, Mo., among others. Tyr-Gly-Gly is not sold in U.S.P. grade, and it is illegal under applicable laws to sell Tyr-Gly-Gly for use as a pharmaceutical. Commercial grade Tyr-Gly-Gly is not considered free of pyrogens, endotoxin, and other pharmaceutically unacceptable constituents. The presence of such pyrogens, endotoxin, and other pharmaceutically unacceptable constituents makes a product unacceptable for use as a drug, as that term is defined by federal statute, both under generally recognized medical principles and under FDA regulations. To the extent of the inventor's knowledge, no pharmaceutical preparations of this product are or have been available.